The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. We demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK controls tumor necrosis factor alpha production and TLR-mediated macrophage responses to Borrelia burgdorferi, the causative agent of Lyme disease, and the TLR1/TLR2-specific agonist PAM(3)CSK(4). JNK1, but not JNK2, activity regulates the expression of the tlr1 gene in the macrophage cell line RAW264.7, as well as in primary CD11b(+) cells. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete.

Protein arginine phosphorylation is a recently discovered modification that affects multiple cellular pathways in Gram-positive bacteria. In particular, the phosphorylation of arginine residues by McsB is critical for regulating the cellular stress response. Given that the highly efficient protein arginine phosphatase YwlE prevents arginine phosphorylation under non-stress conditions, we hypothesized that this enzyme negatively regulates arginine phosphorylation and acts as a sensor of cell stress. To evaluate this hypothesis, we developed the first suite of highly potent and specific SO3-amidine-based YwlE inhibitors. With these protein arginine phosphatase-specific probes, we demonstrated that YwlE activity is suppressed by oxidative stress, which consequently increases arginine phosphorylation, thereby inducing the expression of stress-response genes, which is critical for bacterial virulence. Overall, we predict that these novel chemical tools will be widely used to study the regulation of protein arginine phosphorylation in multiple organisms.